The goal of the proposed research is to better understand the mechanism(s) by which the Polycomb Group (PcG) of proteins mediate gene silencing. This work is significant from a human health perspective because it has the potential to illuminate the mechanism of how PcG proteins participate in cancer and stem cell biology. According to current models of PcG-mediated gene silencing, the methylation of histone H3 by the EZH2 complex serves to recruit the PRC-1 complex as a result of specific interactions between the Polycomb protein (Pc) and methyl H3 lysine 27. While a specific interaction between PC and methylated peptides has been shown, an interaction with methyl K27 in a physiologically relevant context has not been demonstrated. To test the model, specific aim 1 is to address the possibility that PC interacts specifically with methyl H3 K27 in the context of nucleosomes. In the second step of PcG silencing, again according to current models, the ubiquitylation of H2A by the recruited PRC-1 complex leads to the inhibition of transcription. Specific aim 2 is to test this model by establishing an in vitro silencing system that depends on H2A ubiquitylation. [unreadable] [unreadable] [unreadable]